- Email: [email protected]
Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen
ADULT
UROLOGY
ELSEVIER
EFFECT OF CRYOPRESERVATION AND SPERM CONCENTRATION ON LIPID PEROXIDATION IN HUMAN SEMEN Y. WANG,
R. K. SHARMA,
AND
A. AGARWAL
ABSTRACT Objectives. To assess (1) the effect of increased lipid peroxidation on sperm membrane damage that occurs during the freeze-thaw process of cryopreservation; (2) the effect of sperm concentration on lipid peroxidation in normal fertile men; and (3) the relation between sperm motility, morphology, and lipid peroxidation. Methods. Lipid peroxidation was measured as levels of malonaldehyde by the thiobarbituric acid method. Malonaldehyde levels in fresh and frozen-thawed semen specimens from normal donors (n = 20) were compared at a sperm concentration of 20 x 106/mL. Sperm concentrations of 5, 10, 20, and 40 x 106/mL were used to assess the effect of sperm concentration on malonaldehyde levels in frozen specimens. A computerassisted semen analyzer was used to analyze sperm concentrations and motility. Comparison of the effect of TEST-yolk buffer, glycerol, and Ham’s F-l 0 buffer on lipid peroxidation was studied in fresh semen from normal fertile men (n = 5). Results. Malonaldehyde levels did not differ between fresh and cryopreserved semen specimens (P = 0.99). There was no difference in malonaldehyde levels from TEST-yolk buffer, glycerol, and Ham’s F-l 0 buffer. As sperm concentrations increased, so did malonaldehyde levels, from 1.21 ? 0.05 nM/hr at 5 x 106/mL to 3.50 2 0.46 nM/hr at 40 x 1 06/mL (P = 0.000007). Sperm motility, morphology, and malonaldehyde levels in freeze-thaw specimens were not correlated before or after removal of the cryoprotective medium. Conclusions. Malonaldehyde levels do not differ between fresh and frozen semen in normal men. Cryopreservation-induced membrane damage is not related to lipid peroxidation but may be associated with stress induced during freezing and thawing. Malonaldehyde levels increase as sperm concentrations increase. There is no correlation between sperm motility, morphology, and malonaldehyde levels. UROLOGY 50: 409-413, 1997. 0 1997, Elsevier Science Inc. All rights reserved.
ryopreservation of semen specimens has become necessary for assisted reproductive procedures in patients with a wide variety of diagnoses, but poor fertilization and pregnancy rates are seen with cryopreserved semen.l,* Freeze-thaw procedures induce sublethal damage to the sperm plasma membrane, resulting in poor motility and
C
This study was presented in part at the 52nd Annual Meeting of the American Society for Reproductive Medicine, Boston, November 1996. From the Andrology Research and Clinical Laboratories, Department of Urology, Cleveland Clinic Foundation, Cleveland, Ohio Reprint requests: Ashok Agarwaf, Ph.D., Andrology Research and Clinical Laboratories, Department of Urology, A19, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH44195 Submitted: December 20, 1996, accepted (with revisions): March 3, 1997 0 1997, ELSEVIER SCIENCE ALL RIGHTS RESERVED
functional characteristics2 Human spermatozoa are particularly susceptible to lipid peroxidation because cellular and intracellular sperm membranes are rich in polyunsaturated fatty acids.3 Unlike somatic cells that rely on cytoplasmic enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase, for their defense, spermatozoa discharge most of their cytoplasm immediately before spermiation and, as a consequence, lose such protection.4 Lipid peroxidation can cause irreversible loss of sperm motility, and it serves as a biochemical index of semen quality.5,6 The reasons that sperm motion and functional characteristics decrease during the freeze-thaw process are not clear. Cryopreservation-induced sperm membrane damage in normal semen specimens could be caused by lipid peroxidation or membrane stress during the phase transitions that occur during freeze-thaw. The purpose of this
INC. PII
0090-4295/97/$17.00 SOO90-4295(97)00219-7
409
study was to determine (1) the effect of lipid peroxidation on the sperm membrane damage that occurs during the freeze-thaw process; (2) the effect of sperm concentration on lipid peroxidation in normal fertile men; and (3) the relation between sperm motility, morphology, and lipid peroxidation levels. MATERIAL
AND
METHODS
SEMEN COLLECTION AND FREEZING Semen samples from 20 healthy donors were evaluated according to World Health Organization criteria.’ All subjects abstained from ejaculation for 48 hours. The samples were obtained by masturbation into a sterile specimen cup. The ejaculate was allowed to liquefy at 37°C for 30 minutes, and 5 PL of the sample was loaded on a 20-pm counting chamber (Conception Technologies, La Jolla, Calif). Samples were analyzed in a computer-assisted semen analyzer (CASA, Motion Analysis Corporation, Model VP 110, Santa Rosa, Calif). To ensure the accuracy of the CASA results, a manual assessment was done each time, along with the CASA analysis. For cryopreservation of the semen specimens, semen samples were diluted by a dropwise addition of the TEST-yolk buffer freezing medium (Irvine Scientific, Santa Ana, Calif), each time with uniform mixing for 5 minutes. Four additions were carried out to give a final semen with a freezing medium ratio of 1:l (vol/vol). The cryopreservation vials were placed in a -2O’C freezer for 8 minutes and thereafter in liquid nitrogen vapor at -79°C for 2 hours. The vials were then transferred to liquid nitrogen at -196°C. For post-thaw analysis, each semen specimen vial was removed from liquid nitrogen and thawed at room temperature for 5 minutes, followed by incubation for 20 minutes at 37°C.
SPERM MORPHOLOGY DETERMINATION Seminal smears were made from 10 PL of each fresh semen specimen and air dried. Smears were stained with the DiffQuik stain (Baxter Healthcare Corporation, Miami, Fl). Slides were examined by light microscopy at 100X magnification, and at least 100 spermatozoa were counted and classified as normal or abnormal according to the criteria of Menkveld et al.’ Spermatozoa were considered normal when the head was smooth and oval, with a well-defined acrosome comprising 40% to 70% of the sperm head area. Heads that were borderline normal were considered abnormal.
Company) was added. To maintain identical assay volumes, an equal volume of sperm suspension was added to the control samples. The sample was centrifuged at 16OOg for 12 minutes, 1 mL of clear supernatant was collected, and 500 /IL of 2% thiobarbituric acid (Sigma Chemical Company) in 0.2 N NaOH (Sigma Chemical Company) was added to the supernatant. The test tubes were boiled at 100°C for exactly 10 minutes. The sample was cooled in crushed ice, and the optical density was measured at 534 nm with an Ultrospec III spectrophotometer (Pharmacia Biotech Inc., Piscataway, NJ). The malonaldehyde level of the sample was calculated by comparing it with the optical density produced by malonaldehyde standard (Sigma Chemical Company). The lipid peroxidation level was expressed as nM MDA/lo’ sperm/hr.
EFFECT OF TEST-YOLK BUFFER, GLYCEROL, AND HAM’S F-l 0 BUFFER ON LIPID PEROXIDATION The effect of TEST-yolk buffer, glycerol, and Ham’s F-10 buffer on lipid peroxidation was studied in fresh semen specimens from normal fertile men (n = 5). The specimens were divided into three aliquots. Each aliquot was then diluted 1:l with the TEST-yolk buffer freezing medium, 5% glycerol, or Ham’s F-10 buffer (pH 7.4). The samples were then incubated at 37°C for 2 hours. Seminal plasma and the medium were removed by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer. The sperm concentration was adjusted to 20 x I06/mL, and the rest of the lipid peroxidation assay was performed as described above.
EFFECT OF HYDROGEN PEROXIDEON LIPID PEROXIDATION Final concentrations of 0.01% and 0.05% HzOL were used to study the effect of HzOz on lipid peroxidation in fresh semen from normal fertile men (n = 5). Seminal plasma was removed by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer. The sperm concentration was adjusted to 20 x lO“/mL. The specimens were divided into three aliquots of equal volume. One aliquot did not contain HLOz and was used as a control. The second and third aliquots contained the final HzOz concentrations of 0.01% and 0.05%, respectively. All aliquots were incubated at 37°C for 15 minutes. The washed sperm were incubated with 0.25 mL of ferrous sulfate (2.5 mM) and 0.25 mL of sodium ascorbate (12.5 mM) for 15 minutes in a 37°C water bath. The remaining procedure was carried out according to the standard protocol described above.
STATISTICAL ANALYSIS LIPID PEROXIDATION MEASUREMENT Lipid peroxidation levels were measured using the thiobarbituric acid method.‘~” The seminal plasma in fresh semen samples and the cryomedium in cryopreserved semen specimens were removed after thawing by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer (pH 7.4) (Life Technologies Inc., Grand Island, NY). Sperm concentrations of 5, 10, 20, and 40 X 106/mL were used to study the effect of sperm concentration on malonaldehyde levels in frozen semen specimens (n = 10). Ferrous sulfate and sodium ascorbate (Sigma Chemical Company, St. Louis, MO) were used to promote lipid peroxidation. Malonaldehyde levels in fresh and frozen specimens were compared at a sperm concentration of 20 X 106/mL. The washed sperm were incubated with 0.25 mL of ferrous sulfate (2.5 mM) and 0.25 mL of sodium ascorbate (12.5 mM) for 1 hour in a 37°C water bath. Control sperm contained only ferrous sulfate and sodium ascorbate during the incubation period. To precipitate proteins after incubation, 500 PL of 40% ice-cold trichloroacetic acid (Sigma Chemical
410
A paired Student’s t test was used to compare the malonaldehyde levels between fresh and cryopreserved semen. Repeated measures analysis of variance was used to analyze the effect of different freezing media on malonaldehyde levels. Repeated measures analysis of variance on ranks was used to analyze the effect of sperm and HzOz concentrations on malonaldehyde levels; an alpha value of 0.009 with the Bonferroni correction was used in the pairwise sperm concentration comparisons. Pearson’s correlation test was used to assess the correlation between sperm motility, morphology, and malonaldehyde levels. A P value of less than 0.05 was considered statistically significant. Results are expressed as mean value ? standard error or median (interquartile range). The SAS statistical software package (SAS Institute Inc., Cary, NC, 1992) was used for data analysis.
RESULTS Semen analysis [median (interquartile range)] of fresh ejaculates showed a sperm count of 50.6 X UROLOGY
50 C3), 1997
106/mL (37.2, 84.8), motility of 55.8% (50.7%, 62.5%), and morphology of 13% (7%, 17.8%). Malonaldehyde levels did not differ significantly between fresh (24.81 + 1.12 nWIOs sperm/hr) and cryopreserved specimens (24.86 2 1.43 r&I/lo8 sperm/hr) at a sperm concentration of 20 X lo6 sperm/ml. Malonaldehyde levels also did not differ among buffers (P = 0.80): 30.81 2 2.11 nM/ 10’ sperm/hr for TEST-yolk buffer freezing medium, 29.53 t- 1.47 nM/lOs sperm/hr for glycerol, and 29.58 +- 1.20 nM/lO* sperm/hr for Ham’s F10 buffer. Malonaldehyde levels increased linearly with increases in HzOz concentrations. A significant difference was seen in malonaldehyde levels in relation to HzOz concentrations (P = 0.024). Malonaldehyde levels increased from 16.10 -+ 1.29 nM/hr (absence of H,O,) to 19.90 ? 2.23 nM/hr at 0.01% HzOz and 46.00 ? 3.55 nM/hr at 0.05% Hz% Levels of malonaldehyde increased with increases in sperm concentrations, rising from 1.21 + 0.05 nM/hr at 5 X lo6 sperm/ml to 1.62 + 0.12 nM/hr at 10 X lo6 sperm/ml, 2.31 5 0.23 nM/hr at 20 X lo6 sperm/ml, and 3.50 + 0.46 nM/hr at 40 X lo6 sperm/ml (P = 0.000007) (Fig. 1). No significant correlation was seen between sperm motility and malonaldehyde levels in fresh and cryopreserved semen specimens before or after the removal of cryomedium (Fig. 2). Similarly, normal sperm morphology did not correlate with malonaldehyde levels in fresh semen (Y = -0.23; P = 0.36). Similarly, in cryopreserved specimens, sperm morphology did not correlate with malonaldehyde levels (r = 0.16; P = 0.50). COMMENT The thiobarbituric acid reaction for malonaldehyde is the most convenient and widely used assay of lipid peroxidation; the outcome of this assay correlates well with other lipid peroxidation assay methods, including chemiluminescence, pentane or ethane formation, and calorimetric reactions based on the reduction of phospholipid hydroperoxides with potassium iodide.3,” Malonaldehyde is a stable product of lipid peroxidation and therefore can be used as a measure of cumulative lipid peroxidation. 9~10~12 The combination of ferrous sulfate and sodium ascorbate has been used to induce lipid peroxidation as well as to promote the breakdown of lipid peroxides into smaller alkenals like malonaldehyde, the product most often measured in studies involving lipid peroxidation.10,13,‘4 Comparison of lipid peroxidation results among various reports is difficult, mainly because different investigators used various assay methods. 5,9~12,15-18Some variables studied include the UROLOGY
so (31, 1997
4
r
01 0
I I I 10 30 40 20 Sperm concentration (109mL) I
I 50
FIGURE 1. Effect of frozen sperm concentration on mulonaldehyde level. Values represent mean ? SE [n = 10). Malonaldehyde levels differed significantly among different sperm concentrations (P = 0.000007).
type and concentration of lipid peroxidation promoters, duration of sperm incubation, amount of trichloroacetic acid used, centrifugation speed, concentration and amount of thiobarbituric acid used, boiling period, and spectrophotometer wavelength. Our study helps to standardize lipid peroxidation measurement and suggests a normal range of lipid peroxidation in both fresh and frozen semen in normal men. This standardization will make the comparison of malonaldehyde results among different investigators possible. Hydrogen peroxide is the primary toxic reactive oxygen species for human spermatozoa and can induce lipid peroxidation.” In our study, H202 significantly affected malonaldehyde generation in fresh semen, confirming that H202 can also produce malonaldehyde. Investigators have used sperm concentrations ranging from 10 to 200 X 106/mL in determining the level of lipid peroxidation.4’6’20’21 The optimal sperm concentration for lipid peroxidation assay is not known. Although all four different sperm concentrations examined by us can be used to measure malonaldehyde, a concentration of 20 X l@/mL is optimal for studies using a computerassisted semen analyzer because this is the minimum concentration that can be measured with accuracy. Malonaldehyde levels increased linearly with increases in sperm concentration, indicating that sperm generate malonaldehyde.22 The lack of a significant difference in malonaldehyde levels between fresh and frozen semen specimens in normal men suggests that lipid per411
c
0
I
I
I
I
I
I
40
50
60
70
80
90
0
.
l 0
I
I
I
I
I
10
20
30
40
50
Sperm Motility (%) Correlation of sperm motility with malonaldehyde levels and sperm motility showed no correlation (r = 0.16; P = 0.50). FIGURE
2.
lonaldehyde
oxidation is not involved in membrane damage during the freeze-thaw process. The results from our study apply only to semen specimens from normal subjects; however, in subfertile men, higher malonaldehyde levels were observed.23 Sperm motility is highly correlated with fertilization outcome 21., however, after cryopreservation, sperm motility decreases by 25% to 75°b.24 Cryopreservation-induced membrane damage and the decrease in sperm motility are not related to lipid peroxidation but may be linked to membrane stress induced by freezing and thawing. Our findings support earlier reports that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in frozen-thawed human sperm and that the effect of lipid peroxidation on sublethal cryodamage is negligible.25 Our finding of no difference in malonaldehyde levels between cryopreserved and fresh human semen is contrary to previous reports that malonaldehyde levels are higher in cryopreserved semen.26 Malonaldehyde levels may vary as much as lo-fold among individual donors, and considerable variation does occur within different ejaculates from the same subject in fresh semen samples.i5 In our study, the variation in malonaldehyde levels was small (only threefold) among individual donors both in fresh and cryopreserved semen samples; this variation may be due to the different assay methods used by other investigators. In addition to the freeze-thaw process itself, cryopreserved sperm differ from fresh sperm in that they must be placed in a freezing medium 412
levels in fresh [A) and cryopreserved semen (B). Mawith fresh (r = 0.26; P = 0.28) or cryopreserved semen
(TEST-yolk buffer). The buffer used in the assay may induce malonaldehyde generation in human sperm. 27 In normal donors (n = 5), lipid peroxidation did not differ in fresh semen incubated with TEST-yolk buffer compared with Ham’s F-10 buffer or glycerol, indicating that the freezing medium did not contribute to malonaldehyde generation. Malonaldehyde levels show poor correlation with ultramorphologic changes,28 indicating that lipid peroxidation is not due to changes in the subcellular organelles. Others have reported negative and positive correlations between malonaldehyde levels and sperm motility.‘5,25,29 We did not find a significant correlation between sperm motility, normal sperm morphology, and malonaldehyde levels in fresh or cryopreserved semen. This lack of correlation indicates that sperm handling, freezing, and freezing medium used do not cause an increase in the level of lipid peroxidation. Increased levels of lipid peroxidation are not an acquired property of the spermatozoa. CONCLUSIONS Lipid peroxidation in the semen of normal men is not involved in the membrane damage that occurs during cryopreservation. Sperm membrane damage induced during cryopreservation in normal semen specimens may be linked to membrane stress induced by the freeze-thaw process. The lipid peroxidation level increases with an increase in the sperm concentration. Lipid peroxidation UROLOGY
50 (3). 1997
levels are not related to sperm motility morphology.
or normal
ACKNOWLEDGMENT. To Jar-Chi Lee, MS., Department Biostatistics and Epidemiology, Cleveland Clinic Foundation, for help in the statistical analysis of the results.
of
REFERENCES 1. Sherman JK: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil SteriI24: 397-412, 1973. 2. Smith KD, Rodriguez-Rigau LJ, and Steinberger E: The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or cryopreserved semen. Fertil Steri136: 496-502, 1981. 3. Jones R, Mann T, and Sherins R: Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril 31: 531-537, 1979. 4. Irvine DS, and Aitken RJ: Seminal fluid analysis and sperm function testing. Endocrinol Metabol Clin North Am 23:725-748, 1994. 5. Alvarez JG, Touchstone JC, Blasco L, and Storey BT: Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 8: 338-348, 1987. 6. Jones R, Mann T, and Sherins RJ: Adverse effects of peroxidized lipid on human spermatozoa. Proc R Sot Lond 201: 413417, 1978. 7. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (3rd ed). Cambridge, UK, The Press Syndicate of the University of Cambridge, 1992. 8. Menkveld R, Oettle EE, Kruger TF, Swanson RJ, Acosta AA, and Oehninger S (Eds): Atlas of Human Sperm Morphology. Baltimore, Williams & Wilkins, 1991. 9. Hellstrom WJ, Bell M, Wang R, and Sikka SC: Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil Steril 61: 1117-1122, 1994. 10. Bell M, Sikka SC, Rajasekaran M, and Hellstrom WJG: Time course study of hydrogen peroxide induced changes in the lipid peroxidation of human sperm membranes. Assist Reprod Technol Androl 5: 144-150, 1992. 11. Smith MT, Thor H, Hartizell P, and Orrenius S: The measurement of lipid peroxidation in isolated hepatocytes. Biochem Pharmacol31: 19-26, 1982. 12. Hong CY, Lee MF, Lai LJ, and Wang CP: Effect of lipid peroxidation on beating frequency of human sperm tail. Andrologia 26: 61-65, 1994. 13. Aitken RJ, Harkiss D, and Buckingham DW: Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 35: 302-315, 1993.
UROLOGY
50
(3), 1997
14. Alvarez JG, and Storey BT: Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol Reprod 30: 833-841, 1984. 15. Rao B, Soufir JC, Martin M, and David G: Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res 24: 127-134, 1989. 16. Gavella M, and Lipovac V: Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa. Int J Androl 17: 308-313, 1994. 17. Calamera JC, Giovenco P, Quiros MC, Brugo S, Dondero F, and Nicholson RF: Effect of lipid peroxidation upon human spermatic adenosinetriphosphate (ATP): relationship with motility, velocity and linearity of the spermatozoa. Andrologia 21: 48-54, 1989. 18. Aitken RJ, Harkiss D, and Buckingham D: Relationship between iron-catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil 98: 257-265, 1993. 19. de Lamirande E, and Gagnon C: Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod 10 (suppl 1): 1521, 1995. 20. de Lamirande E, and Gagnon C: A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. Int J Androl 16: 21-25, 1993. 21. Richter MA, Haning RV Jr, and Shapiro SS: Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 41: 277-280, 1984. 22. Aitken RJ, Clarkson JS, and Fishel S: Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 41: 183-197, 1989. 23. Wang Y, Sharma RK, Thomas AJ Jr, and Agarwal A: Lipid peroxidation in the semen of subfertile patients (Abstract). Program of the American Urological Association Meeting, New Orleans, 1997, p 181. 24. Bongso TA, Ng SC, Mok H, Lim MN, Teo HL, Wong PC, and Ratnam SS: Effect of sperm motility on human in vitro fertilization. Arch Androl 22: 185-190, 1989. 25. Alvarez JG, and Storey BT: Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J Androl 14: 199-209, 1993. 26. Bell M, Wang R, Hellstrom WJ, and Sikka SC: Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J Androl 14: 472478, 1993. 27. Gomez E, and Aitken J: Impact of in vitro fertilization culture media on peroxidative damage to human spermatozoa. Fertil Steril 65: 880-882, 1996. 28. Geva E, Bartoov B, Zabludovsky V, Lessing JB, LernerGeva L, and Amit A: The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program. Fertil Steril 66: 430434, 1996. 29. Aitken RJ: Pathophysiology of human spermatozoa. Curr Opin Obstet Gynecol6: 128-135, 1994.
413
UROLOGY
ELSEVIER
EFFECT OF CRYOPRESERVATION AND SPERM CONCENTRATION ON LIPID PEROXIDATION IN HUMAN SEMEN Y. WANG,
R. K. SHARMA,
AND
A. AGARWAL
ABSTRACT Objectives. To assess (1) the effect of increased lipid peroxidation on sperm membrane damage that occurs during the freeze-thaw process of cryopreservation; (2) the effect of sperm concentration on lipid peroxidation in normal fertile men; and (3) the relation between sperm motility, morphology, and lipid peroxidation. Methods. Lipid peroxidation was measured as levels of malonaldehyde by the thiobarbituric acid method. Malonaldehyde levels in fresh and frozen-thawed semen specimens from normal donors (n = 20) were compared at a sperm concentration of 20 x 106/mL. Sperm concentrations of 5, 10, 20, and 40 x 106/mL were used to assess the effect of sperm concentration on malonaldehyde levels in frozen specimens. A computerassisted semen analyzer was used to analyze sperm concentrations and motility. Comparison of the effect of TEST-yolk buffer, glycerol, and Ham’s F-l 0 buffer on lipid peroxidation was studied in fresh semen from normal fertile men (n = 5). Results. Malonaldehyde levels did not differ between fresh and cryopreserved semen specimens (P = 0.99). There was no difference in malonaldehyde levels from TEST-yolk buffer, glycerol, and Ham’s F-l 0 buffer. As sperm concentrations increased, so did malonaldehyde levels, from 1.21 ? 0.05 nM/hr at 5 x 106/mL to 3.50 2 0.46 nM/hr at 40 x 1 06/mL (P = 0.000007). Sperm motility, morphology, and malonaldehyde levels in freeze-thaw specimens were not correlated before or after removal of the cryoprotective medium. Conclusions. Malonaldehyde levels do not differ between fresh and frozen semen in normal men. Cryopreservation-induced membrane damage is not related to lipid peroxidation but may be associated with stress induced during freezing and thawing. Malonaldehyde levels increase as sperm concentrations increase. There is no correlation between sperm motility, morphology, and malonaldehyde levels. UROLOGY 50: 409-413, 1997. 0 1997, Elsevier Science Inc. All rights reserved.
ryopreservation of semen specimens has become necessary for assisted reproductive procedures in patients with a wide variety of diagnoses, but poor fertilization and pregnancy rates are seen with cryopreserved semen.l,* Freeze-thaw procedures induce sublethal damage to the sperm plasma membrane, resulting in poor motility and
C
This study was presented in part at the 52nd Annual Meeting of the American Society for Reproductive Medicine, Boston, November 1996. From the Andrology Research and Clinical Laboratories, Department of Urology, Cleveland Clinic Foundation, Cleveland, Ohio Reprint requests: Ashok Agarwaf, Ph.D., Andrology Research and Clinical Laboratories, Department of Urology, A19, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH44195 Submitted: December 20, 1996, accepted (with revisions): March 3, 1997 0 1997, ELSEVIER SCIENCE ALL RIGHTS RESERVED
functional characteristics2 Human spermatozoa are particularly susceptible to lipid peroxidation because cellular and intracellular sperm membranes are rich in polyunsaturated fatty acids.3 Unlike somatic cells that rely on cytoplasmic enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase, for their defense, spermatozoa discharge most of their cytoplasm immediately before spermiation and, as a consequence, lose such protection.4 Lipid peroxidation can cause irreversible loss of sperm motility, and it serves as a biochemical index of semen quality.5,6 The reasons that sperm motion and functional characteristics decrease during the freeze-thaw process are not clear. Cryopreservation-induced sperm membrane damage in normal semen specimens could be caused by lipid peroxidation or membrane stress during the phase transitions that occur during freeze-thaw. The purpose of this
INC. PII
0090-4295/97/$17.00 SOO90-4295(97)00219-7
409
study was to determine (1) the effect of lipid peroxidation on the sperm membrane damage that occurs during the freeze-thaw process; (2) the effect of sperm concentration on lipid peroxidation in normal fertile men; and (3) the relation between sperm motility, morphology, and lipid peroxidation levels. MATERIAL
AND
METHODS
SEMEN COLLECTION AND FREEZING Semen samples from 20 healthy donors were evaluated according to World Health Organization criteria.’ All subjects abstained from ejaculation for 48 hours. The samples were obtained by masturbation into a sterile specimen cup. The ejaculate was allowed to liquefy at 37°C for 30 minutes, and 5 PL of the sample was loaded on a 20-pm counting chamber (Conception Technologies, La Jolla, Calif). Samples were analyzed in a computer-assisted semen analyzer (CASA, Motion Analysis Corporation, Model VP 110, Santa Rosa, Calif). To ensure the accuracy of the CASA results, a manual assessment was done each time, along with the CASA analysis. For cryopreservation of the semen specimens, semen samples were diluted by a dropwise addition of the TEST-yolk buffer freezing medium (Irvine Scientific, Santa Ana, Calif), each time with uniform mixing for 5 minutes. Four additions were carried out to give a final semen with a freezing medium ratio of 1:l (vol/vol). The cryopreservation vials were placed in a -2O’C freezer for 8 minutes and thereafter in liquid nitrogen vapor at -79°C for 2 hours. The vials were then transferred to liquid nitrogen at -196°C. For post-thaw analysis, each semen specimen vial was removed from liquid nitrogen and thawed at room temperature for 5 minutes, followed by incubation for 20 minutes at 37°C.
SPERM MORPHOLOGY DETERMINATION Seminal smears were made from 10 PL of each fresh semen specimen and air dried. Smears were stained with the DiffQuik stain (Baxter Healthcare Corporation, Miami, Fl). Slides were examined by light microscopy at 100X magnification, and at least 100 spermatozoa were counted and classified as normal or abnormal according to the criteria of Menkveld et al.’ Spermatozoa were considered normal when the head was smooth and oval, with a well-defined acrosome comprising 40% to 70% of the sperm head area. Heads that were borderline normal were considered abnormal.
Company) was added. To maintain identical assay volumes, an equal volume of sperm suspension was added to the control samples. The sample was centrifuged at 16OOg for 12 minutes, 1 mL of clear supernatant was collected, and 500 /IL of 2% thiobarbituric acid (Sigma Chemical Company) in 0.2 N NaOH (Sigma Chemical Company) was added to the supernatant. The test tubes were boiled at 100°C for exactly 10 minutes. The sample was cooled in crushed ice, and the optical density was measured at 534 nm with an Ultrospec III spectrophotometer (Pharmacia Biotech Inc., Piscataway, NJ). The malonaldehyde level of the sample was calculated by comparing it with the optical density produced by malonaldehyde standard (Sigma Chemical Company). The lipid peroxidation level was expressed as nM MDA/lo’ sperm/hr.
EFFECT OF TEST-YOLK BUFFER, GLYCEROL, AND HAM’S F-l 0 BUFFER ON LIPID PEROXIDATION The effect of TEST-yolk buffer, glycerol, and Ham’s F-10 buffer on lipid peroxidation was studied in fresh semen specimens from normal fertile men (n = 5). The specimens were divided into three aliquots. Each aliquot was then diluted 1:l with the TEST-yolk buffer freezing medium, 5% glycerol, or Ham’s F-10 buffer (pH 7.4). The samples were then incubated at 37°C for 2 hours. Seminal plasma and the medium were removed by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer. The sperm concentration was adjusted to 20 x I06/mL, and the rest of the lipid peroxidation assay was performed as described above.
EFFECT OF HYDROGEN PEROXIDEON LIPID PEROXIDATION Final concentrations of 0.01% and 0.05% HzOL were used to study the effect of HzOz on lipid peroxidation in fresh semen from normal fertile men (n = 5). Seminal plasma was removed by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer. The sperm concentration was adjusted to 20 x lO“/mL. The specimens were divided into three aliquots of equal volume. One aliquot did not contain HLOz and was used as a control. The second and third aliquots contained the final HzOz concentrations of 0.01% and 0.05%, respectively. All aliquots were incubated at 37°C for 15 minutes. The washed sperm were incubated with 0.25 mL of ferrous sulfate (2.5 mM) and 0.25 mL of sodium ascorbate (12.5 mM) for 15 minutes in a 37°C water bath. The remaining procedure was carried out according to the standard protocol described above.
STATISTICAL ANALYSIS LIPID PEROXIDATION MEASUREMENT Lipid peroxidation levels were measured using the thiobarbituric acid method.‘~” The seminal plasma in fresh semen samples and the cryomedium in cryopreserved semen specimens were removed after thawing by centrifugation at 300g for 7 minutes, followed by washing in Ham’s F-10 buffer (pH 7.4) (Life Technologies Inc., Grand Island, NY). Sperm concentrations of 5, 10, 20, and 40 X 106/mL were used to study the effect of sperm concentration on malonaldehyde levels in frozen semen specimens (n = 10). Ferrous sulfate and sodium ascorbate (Sigma Chemical Company, St. Louis, MO) were used to promote lipid peroxidation. Malonaldehyde levels in fresh and frozen specimens were compared at a sperm concentration of 20 X 106/mL. The washed sperm were incubated with 0.25 mL of ferrous sulfate (2.5 mM) and 0.25 mL of sodium ascorbate (12.5 mM) for 1 hour in a 37°C water bath. Control sperm contained only ferrous sulfate and sodium ascorbate during the incubation period. To precipitate proteins after incubation, 500 PL of 40% ice-cold trichloroacetic acid (Sigma Chemical
410
A paired Student’s t test was used to compare the malonaldehyde levels between fresh and cryopreserved semen. Repeated measures analysis of variance was used to analyze the effect of different freezing media on malonaldehyde levels. Repeated measures analysis of variance on ranks was used to analyze the effect of sperm and HzOz concentrations on malonaldehyde levels; an alpha value of 0.009 with the Bonferroni correction was used in the pairwise sperm concentration comparisons. Pearson’s correlation test was used to assess the correlation between sperm motility, morphology, and malonaldehyde levels. A P value of less than 0.05 was considered statistically significant. Results are expressed as mean value ? standard error or median (interquartile range). The SAS statistical software package (SAS Institute Inc., Cary, NC, 1992) was used for data analysis.
RESULTS Semen analysis [median (interquartile range)] of fresh ejaculates showed a sperm count of 50.6 X UROLOGY
50 C3), 1997
106/mL (37.2, 84.8), motility of 55.8% (50.7%, 62.5%), and morphology of 13% (7%, 17.8%). Malonaldehyde levels did not differ significantly between fresh (24.81 + 1.12 nWIOs sperm/hr) and cryopreserved specimens (24.86 2 1.43 r&I/lo8 sperm/hr) at a sperm concentration of 20 X lo6 sperm/ml. Malonaldehyde levels also did not differ among buffers (P = 0.80): 30.81 2 2.11 nM/ 10’ sperm/hr for TEST-yolk buffer freezing medium, 29.53 t- 1.47 nM/lOs sperm/hr for glycerol, and 29.58 +- 1.20 nM/lO* sperm/hr for Ham’s F10 buffer. Malonaldehyde levels increased linearly with increases in HzOz concentrations. A significant difference was seen in malonaldehyde levels in relation to HzOz concentrations (P = 0.024). Malonaldehyde levels increased from 16.10 -+ 1.29 nM/hr (absence of H,O,) to 19.90 ? 2.23 nM/hr at 0.01% HzOz and 46.00 ? 3.55 nM/hr at 0.05% Hz% Levels of malonaldehyde increased with increases in sperm concentrations, rising from 1.21 + 0.05 nM/hr at 5 X lo6 sperm/ml to 1.62 + 0.12 nM/hr at 10 X lo6 sperm/ml, 2.31 5 0.23 nM/hr at 20 X lo6 sperm/ml, and 3.50 + 0.46 nM/hr at 40 X lo6 sperm/ml (P = 0.000007) (Fig. 1). No significant correlation was seen between sperm motility and malonaldehyde levels in fresh and cryopreserved semen specimens before or after the removal of cryomedium (Fig. 2). Similarly, normal sperm morphology did not correlate with malonaldehyde levels in fresh semen (Y = -0.23; P = 0.36). Similarly, in cryopreserved specimens, sperm morphology did not correlate with malonaldehyde levels (r = 0.16; P = 0.50). COMMENT The thiobarbituric acid reaction for malonaldehyde is the most convenient and widely used assay of lipid peroxidation; the outcome of this assay correlates well with other lipid peroxidation assay methods, including chemiluminescence, pentane or ethane formation, and calorimetric reactions based on the reduction of phospholipid hydroperoxides with potassium iodide.3,” Malonaldehyde is a stable product of lipid peroxidation and therefore can be used as a measure of cumulative lipid peroxidation. 9~10~12 The combination of ferrous sulfate and sodium ascorbate has been used to induce lipid peroxidation as well as to promote the breakdown of lipid peroxides into smaller alkenals like malonaldehyde, the product most often measured in studies involving lipid peroxidation.10,13,‘4 Comparison of lipid peroxidation results among various reports is difficult, mainly because different investigators used various assay methods. 5,9~12,15-18Some variables studied include the UROLOGY
so (31, 1997
4
r
01 0
I I I 10 30 40 20 Sperm concentration (109mL) I
I 50
FIGURE 1. Effect of frozen sperm concentration on mulonaldehyde level. Values represent mean ? SE [n = 10). Malonaldehyde levels differed significantly among different sperm concentrations (P = 0.000007).
type and concentration of lipid peroxidation promoters, duration of sperm incubation, amount of trichloroacetic acid used, centrifugation speed, concentration and amount of thiobarbituric acid used, boiling period, and spectrophotometer wavelength. Our study helps to standardize lipid peroxidation measurement and suggests a normal range of lipid peroxidation in both fresh and frozen semen in normal men. This standardization will make the comparison of malonaldehyde results among different investigators possible. Hydrogen peroxide is the primary toxic reactive oxygen species for human spermatozoa and can induce lipid peroxidation.” In our study, H202 significantly affected malonaldehyde generation in fresh semen, confirming that H202 can also produce malonaldehyde. Investigators have used sperm concentrations ranging from 10 to 200 X 106/mL in determining the level of lipid peroxidation.4’6’20’21 The optimal sperm concentration for lipid peroxidation assay is not known. Although all four different sperm concentrations examined by us can be used to measure malonaldehyde, a concentration of 20 X l@/mL is optimal for studies using a computerassisted semen analyzer because this is the minimum concentration that can be measured with accuracy. Malonaldehyde levels increased linearly with increases in sperm concentration, indicating that sperm generate malonaldehyde.22 The lack of a significant difference in malonaldehyde levels between fresh and frozen semen specimens in normal men suggests that lipid per411
c
0
I
I
I
I
I
I
40
50
60
70
80
90
0
.
l 0
I
I
I
I
I
10
20
30
40
50
Sperm Motility (%) Correlation of sperm motility with malonaldehyde levels and sperm motility showed no correlation (r = 0.16; P = 0.50). FIGURE
2.
lonaldehyde
oxidation is not involved in membrane damage during the freeze-thaw process. The results from our study apply only to semen specimens from normal subjects; however, in subfertile men, higher malonaldehyde levels were observed.23 Sperm motility is highly correlated with fertilization outcome 21., however, after cryopreservation, sperm motility decreases by 25% to 75°b.24 Cryopreservation-induced membrane damage and the decrease in sperm motility are not related to lipid peroxidation but may be linked to membrane stress induced by freezing and thawing. Our findings support earlier reports that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in frozen-thawed human sperm and that the effect of lipid peroxidation on sublethal cryodamage is negligible.25 Our finding of no difference in malonaldehyde levels between cryopreserved and fresh human semen is contrary to previous reports that malonaldehyde levels are higher in cryopreserved semen.26 Malonaldehyde levels may vary as much as lo-fold among individual donors, and considerable variation does occur within different ejaculates from the same subject in fresh semen samples.i5 In our study, the variation in malonaldehyde levels was small (only threefold) among individual donors both in fresh and cryopreserved semen samples; this variation may be due to the different assay methods used by other investigators. In addition to the freeze-thaw process itself, cryopreserved sperm differ from fresh sperm in that they must be placed in a freezing medium 412
levels in fresh [A) and cryopreserved semen (B). Mawith fresh (r = 0.26; P = 0.28) or cryopreserved semen
(TEST-yolk buffer). The buffer used in the assay may induce malonaldehyde generation in human sperm. 27 In normal donors (n = 5), lipid peroxidation did not differ in fresh semen incubated with TEST-yolk buffer compared with Ham’s F-10 buffer or glycerol, indicating that the freezing medium did not contribute to malonaldehyde generation. Malonaldehyde levels show poor correlation with ultramorphologic changes,28 indicating that lipid peroxidation is not due to changes in the subcellular organelles. Others have reported negative and positive correlations between malonaldehyde levels and sperm motility.‘5,25,29 We did not find a significant correlation between sperm motility, normal sperm morphology, and malonaldehyde levels in fresh or cryopreserved semen. This lack of correlation indicates that sperm handling, freezing, and freezing medium used do not cause an increase in the level of lipid peroxidation. Increased levels of lipid peroxidation are not an acquired property of the spermatozoa. CONCLUSIONS Lipid peroxidation in the semen of normal men is not involved in the membrane damage that occurs during cryopreservation. Sperm membrane damage induced during cryopreservation in normal semen specimens may be linked to membrane stress induced by the freeze-thaw process. The lipid peroxidation level increases with an increase in the sperm concentration. Lipid peroxidation UROLOGY
50 (3). 1997
levels are not related to sperm motility morphology.
or normal
ACKNOWLEDGMENT. To Jar-Chi Lee, MS., Department Biostatistics and Epidemiology, Cleveland Clinic Foundation, for help in the statistical analysis of the results.
of
REFERENCES 1. Sherman JK: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil SteriI24: 397-412, 1973. 2. Smith KD, Rodriguez-Rigau LJ, and Steinberger E: The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or cryopreserved semen. Fertil Steri136: 496-502, 1981. 3. Jones R, Mann T, and Sherins R: Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril 31: 531-537, 1979. 4. Irvine DS, and Aitken RJ: Seminal fluid analysis and sperm function testing. Endocrinol Metabol Clin North Am 23:725-748, 1994. 5. Alvarez JG, Touchstone JC, Blasco L, and Storey BT: Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 8: 338-348, 1987. 6. Jones R, Mann T, and Sherins RJ: Adverse effects of peroxidized lipid on human spermatozoa. Proc R Sot Lond 201: 413417, 1978. 7. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (3rd ed). Cambridge, UK, The Press Syndicate of the University of Cambridge, 1992. 8. Menkveld R, Oettle EE, Kruger TF, Swanson RJ, Acosta AA, and Oehninger S (Eds): Atlas of Human Sperm Morphology. Baltimore, Williams & Wilkins, 1991. 9. Hellstrom WJ, Bell M, Wang R, and Sikka SC: Effect of sodium nitroprusside on sperm motility, viability, and lipid peroxidation. Fertil Steril 61: 1117-1122, 1994. 10. Bell M, Sikka SC, Rajasekaran M, and Hellstrom WJG: Time course study of hydrogen peroxide induced changes in the lipid peroxidation of human sperm membranes. Assist Reprod Technol Androl 5: 144-150, 1992. 11. Smith MT, Thor H, Hartizell P, and Orrenius S: The measurement of lipid peroxidation in isolated hepatocytes. Biochem Pharmacol31: 19-26, 1982. 12. Hong CY, Lee MF, Lai LJ, and Wang CP: Effect of lipid peroxidation on beating frequency of human sperm tail. Andrologia 26: 61-65, 1994. 13. Aitken RJ, Harkiss D, and Buckingham DW: Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 35: 302-315, 1993.
UROLOGY
50
(3), 1997
14. Alvarez JG, and Storey BT: Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol Reprod 30: 833-841, 1984. 15. Rao B, Soufir JC, Martin M, and David G: Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res 24: 127-134, 1989. 16. Gavella M, and Lipovac V: Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa. Int J Androl 17: 308-313, 1994. 17. Calamera JC, Giovenco P, Quiros MC, Brugo S, Dondero F, and Nicholson RF: Effect of lipid peroxidation upon human spermatic adenosinetriphosphate (ATP): relationship with motility, velocity and linearity of the spermatozoa. Andrologia 21: 48-54, 1989. 18. Aitken RJ, Harkiss D, and Buckingham D: Relationship between iron-catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil 98: 257-265, 1993. 19. de Lamirande E, and Gagnon C: Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod 10 (suppl 1): 1521, 1995. 20. de Lamirande E, and Gagnon C: A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. Int J Androl 16: 21-25, 1993. 21. Richter MA, Haning RV Jr, and Shapiro SS: Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 41: 277-280, 1984. 22. Aitken RJ, Clarkson JS, and Fishel S: Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 41: 183-197, 1989. 23. Wang Y, Sharma RK, Thomas AJ Jr, and Agarwal A: Lipid peroxidation in the semen of subfertile patients (Abstract). Program of the American Urological Association Meeting, New Orleans, 1997, p 181. 24. Bongso TA, Ng SC, Mok H, Lim MN, Teo HL, Wong PC, and Ratnam SS: Effect of sperm motility on human in vitro fertilization. Arch Androl 22: 185-190, 1989. 25. Alvarez JG, and Storey BT: Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J Androl 14: 199-209, 1993. 26. Bell M, Wang R, Hellstrom WJ, and Sikka SC: Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J Androl 14: 472478, 1993. 27. Gomez E, and Aitken J: Impact of in vitro fertilization culture media on peroxidative damage to human spermatozoa. Fertil Steril 65: 880-882, 1996. 28. Geva E, Bartoov B, Zabludovsky V, Lessing JB, LernerGeva L, and Amit A: The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program. Fertil Steril 66: 430434, 1996. 29. Aitken RJ: Pathophysiology of human spermatozoa. Curr Opin Obstet Gynecol6: 128-135, 1994.
413